(i) Field of the Invention
The present invention relates to regulation of genes expressed during seed maturation and, more particularly, to proteins (and corresponding DNAs) which bind with high affinity to promoter regions of genes expressed during seed maturation. The invention further relates to use of such proteins in order to enhance or reduce the expression of seed storage protein and oil-body protein genes in transgenic plants, thereby altering the protein and oil composition of the seeds.
(ii) Description of Related Art
Genes expressed specifically during seed maturation possess some of the most highly regulated promoters found in higher vascular plants. The main maturation associated products of the common bean are 7S globulin storage proteins (phaseolins) and lectins, which together represent 80% of all seed proteins.
One of the most distinctive characteristics of seed storage protein (SSP) and lectin genes and their promoters is that they are most active during seed maturation and become rapidly repressed at the time of seed abscission (Hughes & Galau, 1989; Murray & Kennard, 1984). This decreased expression of SSP and lectin genes towards the end of seed maturation distinguishes them from late embryogenesis abundant (Lea) genes which continue to be expressed throughout seed abscission (Dure. 1985). More than a decade of research into the structure and function of SSP and lectin promoters has yielded a few DNA motifs implicated in tissue-specificity and abscisic acid (ABA) inducibility (Thomas, 1993). The most abundant DNA binding activities found in developing seeds interact with AT-rich and other, apparently non-essential sequences (Jofuku et al., 1987; Bustos et al., 1991; Fujiwara & Beachy, 1994). No significant DNA binding activities have been found to interact with RY repeats of 7S and 11S SSP promoters shown to be necessary for maturation induction of those promoters in vivo (Baumlein et al., 1992; Lelievre et al., 1992; Chamberland et al., 1992; Fujiwara & Beachy, 1994). Consequently, despite considerable efforts in that direction, cloning of maturation regulatory trans-acting factors remains an unfulfilled goal.
Except for Brassica napus, Arabidopsis thaliana or the legumes, the only plant species in which the molecular biology of seed maturation has been investigated in detail are the cereals, mainly wheat, rice and maize. Loss-of-function mutations at the maize Opaque2 locus are associated with an 80% reduction in the synthesis of 22 kDa zeins in the endosperm of corn kernels (Motto et al., 1988). The Opaque2 gene (Schmidt et al., 1990) encodes a transcription factor that binds to and activates the promoters of 22 kDa zein (Schmidt et al., 1992; Yunes et al 1994) and 32b (Lohmer et al., 1991 ) genes, but has little effect on the expression of 19 kDa zein genes. Recombinant Opaque2 protein expressed as a fusion with E. coli galactosidase binds to the sequence [SEQ ID NO.:1] .sup.5' CACACGTCAA.sup.3' of the .delta.-phaseolin promoter; more importantly, nuclear factors present in immature bean cotyledons also bind to this motif, and display the same apparent sequence specificity as Opaque2 (Bustos et al., submitted), suggesting that Opaque2-like proteins may be involved in phaseolin regulation. Opaque2 belongs to the family of basic-leucine zipper (bZIP) transcription factors. Plant bZIP factors form a heterogeneous family of proteins that commonly bind to DNA sequences containing a .sup.5' ACGT.sup.3' core (Weisshaar et al., 1991; Foster et al., 1994). The bZIP domain consists of a basic region and an amphipathic .alpha.-helical segment containing three or more heptad repeats of leucine residues (leucine zipper). The basic region contacts the DNA double-helix, and the leucine zipper functions as a dimerization domain.
From the above discussion, it should be apparent that regulation of genes expressed during seed maturation has yet to be achieved given the failure of the art to identify and characterize those proteins serving such a regulatory function. Indeed, it has been found that in most agronomically important plants, seed storage proteins and oil-body proteins are encoded by "gene families," i.e., sets of a few to hundreds of genes. In order to effect significant changes in seed protein or oil compositions, therefore, it is necessary to simultaneously alter the activity of potentially large numbers of genes at once. This to date has presented an obstacle to genetic engineering of seed crops. For this reason, the numerous important commercial implications stemming from control of this regulatory function, such as control of the oil and protein content of a seed, have not been realized.